For the past century, environmental engineers have been using the activated sludge system and anaerobic digestion to successfully treat municipal wastewater (Metcalf & Eddy (2003). Wastewater Engineering: Treatment and Reuse. New York, N.Y., McGraw-Hill). In the United States, the nitrification process in the activated sludge system of public owned treatment works (POTWs) is very expensive with capital costs of the aeration basin alone valued at $26B and annual electricity costs of $335M (EPA (1996). Clean Watersheds Needs Survey (CWNS). W.D.C. Office of Water; Goldstein, R. and W. Smith (2002). Water & Sustainability (Volume 4): U.S. Electricity Consumption for Water Supply & Treatment—The Next Half Century. Palo Alto, EPRI; EPA (2007). Biological Removal Processes and Costs. W.D.C. Office of Water). More recently, the United States Environmental Protection Agency has proposed a stricter effluent nutrient limit (Total N of 0.82-1.73 mg/L and Total P of 0.069-0.415 mg/L) for Florida POTWs that is estimated to cost $24.4-50.7B in capital investment and increase annual operating expenses by $0.4-1.3B (Oskowis, J. (2009). Re: Numeric Nutrient Criteria Cost Implications for Florida POTW's G. C. Crist). Over half of the capital investment will upgrade the existing activated sludge system to an advanced biological nutrient removal (BNR) system. It is anticipated that the numerous extended aeration plants used for secondary treatment of wastewater in FL will be upgraded to BP5 or other advanced BNR system. The BP5 upgrade of the extended aeration plants will most likely not include the introduction of a primary clarifier and instead, the process will be operated with an elevated IRR. The impact of the IRR on the microbial ecology of BNR systems has not been reported.
Researchers used empirical studies to identify key operating parameters for these systems to ensure effective performance. In order to increase the protection of surface waters from excessive oxygen demand from treated wastewater, engineers have modified the simple aeration basin to include anoxic and anaerobic zones and recycled nitrate-rich wastewater and anaerobic digester supernatant to promote biological nutrient removal. For biological nitrogen removal, the operation of the conventional activated sludge system was modified to include a nitrification step for the biological conversion of ammonium to nitrate, which is subsequently removed via denitrification to nitrogen gas by an anoxic zone in the activated sludge system or fixed-film denitrification process of the secondary clarifier effluent. These activated sludge system designs and operations are dependent on providing adequate biomass concentration in the aeration tank, environmental conditions for the biomass, and adequate time for the bioreaction. Carbonaceous biochemical oxygen demand (BOD) and ammonium are consumed as substrate by the heterotrophic and nitrifying bacteria, respectively. The heterotrophic bacteria grow much faster than the nitrifying bacteria, which handicap the current activated sludge system. Because the overall reaction rate (r) for BOD or ammonium is directly related to the product of the specific growth rate (μ) and biomass concentration (X), the nitrification rate requires much higher biomass concentration to compensate for the much lower specific growth rate compared to the heterotrophic bacteria.
The conventional nitrification process requires a lengthy solids retention time (SRT), which prevents the washout of the slow-growing nitrifying bacteria and provides sufficient biomass to ensure adequate bulk nitrification rates. The biomass concentration of both heterotrophic and nitrifying bacteria is directly related to the wasting rate of the settled sludge from the secondary clarifier, with SRT increasing as the wasting rate decreases. To achieve complete nitrification (ammonium oxidation to nitrate), the activated sludge system is typically operated at an elevated SRT of 8 days or more. This SRT ensures an adequate concentration of nitrifying bacteria, but also results in the accumulation of the heterotrophic bacteria. While this approach has been successful in treating both BOD and Ammonium, the aeration basin is not efficient.
The internal recycle of the nitrate-rich, treated wastewater to an anoxic basin or zone is commonly used in suspended growth systems to reduce the nitrate concentration of treated wastewater in subsequent treatment basins or the secondary clarifier (Metcalf & Eddy (2003). Wastewater Engineering: Treatment and Reuse. New York, N.Y., McGraw-Hill). High internal recycle rates (IRR) above 400% offer minimal improvement with respect to nitrate concentration and may cause aerobic conditions in the anoxic basin. However, in the Tampa metro region, four public owned treatment works (POTWs) that utilize the 5-stage BardenPho (BP5) process for nitrogen and phosphorus removal, reported high IRR of 545-806% (personal communication). This high IRR for this type of BNR system far exceeds guidelines, but is most likely due the lack of a primary clarifier in these systems (Metcalf & Eddy (2003). Wastewater Engineering: Treatment and Reuse. New York, N.Y., McGraw-Hill). The additional primary solids entering the fermentation stage provides both fermentative bacteria and organic substrate for the generation of volatile fatty acids, which is necessary for phosphorus accumulating organisms (PAO). However, the primary solids require additional aerobic treatment. In essence, the high IRR results in a hybrid BNR system that is both BP5 system and aerobic digester, which is possible by increasing the IRR. All four POTWs that employ the high IRR report excellent nitrogen and phosphorus removal.
With the discovery of the phylogenetic tree of life, the major microbial populations were identified in biological reactor systems using mature molecular biology tools, such as membrane hybridizations and fluorescence in situ hybridizations (FISH) (Amann, R. I., W. Ludwig, et al. (1995). “Phylogenetic identification and in situ detection of individual microbial cells without cultivation.” Microbiol Rev 59(1): 143-69). Recently, molecular biology tools have been developed and successfully utilized to determine the microbial community structure and function in these systems. The investigation of the microbiology of biological reactor systems consists of determining the identity and abundance of microbes present (microbial community structure) and their role in the activated sludge process (microbial community function). Traditionally, light microscopy or culture-based methods were used to characterize the microbial structure of biological reactor systems. More recently, new tools that draw on molecular biology and a new view of the phylogeny of life have been developed to identity bacteria and determine their function.
The nitrification process is an example of a well-studied process, whose fundamental knowledge of the microbial ecology is still evolving. For years, the practicing engineer was taught that the process was dominated by species of the genera Nitrosomonas and Nitrobacter, which represent the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB), respectively. The diversity of recognized species of AOB was evaluated by both 16S rRNA and amoA sequence information and three major groups were identified: (1) nitrosomonads, which has five distinct clusters, (2) Nitrosospira species, and (3) Nitrosococcus species (Purkhold, U., A. Pommerening-Roser, et al. (2000). “Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys.” Appl Environ Microbiol 66(12): 5368-82). The nitrosomonads were the dominant AOB in all but two of eleven wastewater treatment plants evaluated in this study. More recently, this fundamental knowledge of the nitrification process was updated by the findings from the application of molecular biology based methods, which determined that the predominant AOB are nitrosomonads and Nitrosospira, while the dominant NOB is Nitrospira (Juretschko, S., G. Timmermann, et al. (1998). “Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations.” Appl Environ Microbiol 64(8): 3042-51; Burrell, P., J. Keller, et al. (1999). “Characterisation of the bacterial consortium involved in nitrite oxidation in activated sludge.” Water Science and Technology 39(6): 45-52; Aoi, Y., T. Miyoshi, et al. (2000). “Microbial ecology of nitrifying bacteria in wastewater treatment process examined by fluorescence in situ hybridization.” J Biosci Bioeng 90(3): 234-40; Coskuner, G. and T. P. Curtis (2002). “In situ characterization of nitrifiers in an activated sludge plant: detection of Nitrobacter Spp.” J Appl Microbiol 93(3): 431-7; Dionisi, H. M., A. C. Layton, et al. (2002). “Quantification of Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR.” Appl Environ Microbiol 68(1): 245-53; Harms, G., A. C. Layton, et al. (2003). “Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant.” Environ Sci Technol 37(2): 343-51). A more recent study of seven full-scale wastewater treatment plants revealed that nitrosomonads and Nitrosospira, and Nitrobacter and Nitrospira were the dominant AOB and NOB, respectively (Siripong, S, and B. E. Rittmann (2007). “Diversity study of nitrifying bacteria in full-scale municipal wastewater treatment plants.” Water Research 41(5): 1110-1120). The diversity of the nitrifying bacteria was very similar across the seven plants; however, seasonal temperature variation was identified as a cause of changes in diversity. The authors suggested that the coexistence of these nitrifiers is evidence of functional redundancy, which assists in maintaining performance stability. Beyond nitrification, Nitrosomonas strains have demonstrated the capability to denitrify (Schmidt, I., O, Sliekers, et al. (2003). “New concepts of microbial treatment processes for the nitrogen removal in wastewater.” Fems Microbiology Reviews 27(4): 481-492; Milner, M. G., T. P. Curtis, et al. (2008). “Presence and activity of ammonia-oxidising bacteria detected amongst the overall bacterial diversity along a physico-chemical gradient of a nitrifying wastewater treatment plant.” Water Research 42(12): 2863-2872).
However, current wastewater technology does not separate the SRT of nitrifying from other bacteria, and therefore inefficiently uses bacteria to aid in wastewater treatment. Accordingly, the present invention provides methods to uncouple the SRT of the nitrifying bacteria and other microorganism of interest from the SRT of the remaining bacteria to enhance nitrification or other metabolic functions necessary for efficient biological treatment of wastewater.